DataSheet2_Isoquercitrin alleviates pirarubicin-induced cardiotoxicity in vivo and in vitro by inhibiting apoptosis through Phlpp1/AKT/Bcl-2 signaling pathway.docx

Introduction: Due to the cardiotoxicity of pirarubicin (THP), it is necessary to investigate new compounds for the treatment of THP-induced cardiotoxicity. Isoquercitrin (IQC) is a natural flavonoid with anti-oxidant and anti-apoptosis properties. Thus, the present study aimed to investigate the influence of IQC on preventing the THP-induced cardiotoxicity in vivo and in vitro.
Methods: The optimal concentration and time required for IQC to prevent THP-induced cardiomyocyte damage were determined by an MTT assay. The protective effect was further verified in H9c2 and HCM cells using dichlorodihydrofluorescein diacetate fluorescent probes, MitoTracker Red probe, enzyme-linked immunosorbent assay, JC-1 probe, and real time-quantitative polymerase chain reaction (RT-qPCR). Rats were administered THP to establish cardiotoxicity. An electrocardiogram (ECG) was performed, and cardiac hemodynamics, myocardial enzymes, oxidative stress indicators, and hematoxylin-eosin staining were studied. Voltage-dependent anion channel 1 (VDAC1), adenine nucleotide translocase 1 (ANT1), and cyclophilin D (CYPD) were detected by qRT-PCR, and the Phlpp1/AKT/Bcl-2 axis proteins were detected by western blot, confirming that IQC markedly increased cell viability and superoxide dismutase (SOD) levels, diminished the levels of ROS and MDA, and elevated mitochondrial function and apoptosis in vivo and in vitro.
Results: Results showed that IQC reduced THP-induced myocardial histopathological injury, electrocardiogram (ECG) abnormalities, and cardiac dysfunction in vivo. IQC also decreased serum levels of MDA, BNP, CK-MB, c-TnT, and LDH, while increasing levels of SOD and GSH. We also found that IQC significantly reduced VDAC1, ANT1, and CYPD mRNA expression. In addition, IQC controlled apoptosis by modulating Phlpp1/AKT/Bcl-2 signaling pathways. IQC markedly increased H9c2 and HCM cell viability and SOD levels, diminished the levels of ROS and MDA, and elevated mitochondrial function in H9c2 and HCM cells to defend against THP-induced cardiomyocyte apoptosis in vitro. The AKT inhibitor IMQ demonstrated that IQC lacked antioxidant and anti-apoptotic properties. Moreover, our data showed that IQC regulates Phlpp1 expression, thereby influencing the expression levels of p-AKT, cytochrome c, caspase-3, caspase-9, Bcl-2, and Bax.
Discussion: In conclusion, our results indicate that IQC protects the changes in mitochondrial membrane permeability in cardiomyocytes by regulating the Phlpp1/AKT/Bcl-2 signaling pathway, inhibits the release of cytc from the mitochondrial inner membrane to the cytoplasm, forms apoptotic bodies, induces cell apoptosis, and reduces THP induced cardiotoxicity.

引言：鉴于吡柔比星（pirarubicin, THP）具有心脏毒性，亟需探索可用于治疗THP诱导性心脏毒性的新型化合物。异槲皮苷（Isoquercitrin, IQC）是一类兼具抗氧化与抗凋亡特性的天然黄酮类化合物。本研究旨在探究IQC在体内及体外模型中对THP诱导性心脏毒性的预防作用。
方法：本研究通过MTT比色法（MTT assay）确定了IQC预防THP诱导心肌细胞损伤所需的最优浓度与作用时间。进一步采用二氯二氢荧光素二乙酸酯（dichlorodihydrofluorescein diacetate）荧光探针、MitoTracker Red探针、酶联免疫吸附实验（enzyme-linked immunosorbent assay）、JC-1探针以及实时定量聚合酶链反应（real time-quantitative polymerase chain reaction, RT-qPCR），在H9c2细胞与HCM细胞中验证了其保护作用。通过给大鼠注射THP构建心脏毒性模型，随后进行心电图（electrocardiogram, ECG）检测，分析心脏血流动力学指标、心肌酶谱、氧化应激标志物，并开展苏木精-伊红染色（hematoxylin-eosin staining）观察。采用qRT-PCR检测电压依赖性阴离子通道1（voltage-dependent anion channel 1, VDAC1）、腺嘌呤核苷酸转运体1（adenine nucleotide translocase 1, ANT1）和亲环素D（cyclophilin D, CYPD）的mRNA表达水平，通过蛋白质印迹（western blot）检测Phlpp1/AKT/Bcl-2信号轴蛋白表达，结果证实IQC可显著提升细胞存活率与超氧化物歧化酶（superoxide dismutase, SOD）活性，降低活性氧（reactive oxygen species, ROS）与丙二醛（malondialdehyde, MDA）水平，改善体内外模型的线粒体功能并抑制细胞凋亡。
结果：体内实验结果表明，IQC可减轻THP诱导的心肌组织病理损伤、心电图异常与心脏功能障碍。IQC可降低血清中MDA、脑钠肽（brain natriuretic peptide, BNP）、肌酸激酶同工酶MB（creatine kinase isoenzyme MB, CK-MB）、心肌肌钙蛋白T（cardiac troponin T, c-TnT）以及乳酸脱氢酶（lactate dehydrogenase, LDH）的水平，同时提升SOD与谷胱甘肽（glutathione, GSH）的含量。本研究还发现，IQC可显著下调VDAC1、ANT1与CYPD的mRNA表达水平。此外，IQC通过调控Phlpp1/AKT/Bcl-2信号通路抑制细胞凋亡。体外实验中，IQC可显著提升H9c2细胞与HCM细胞的存活率及SOD活性，降低ROS与MDA水平，改善线粒体功能，从而对抗THP诱导的心肌细胞凋亡。通过AKT抑制剂IMQ干预实验证实，IQC的抗氧化与抗凋亡作用依赖于该通路。本研究数据进一步显示，IQC可调控Phlpp1的表达，进而影响磷酸化AKT（phosphorylated AKT, p-AKT）、细胞色素c（cytochrome c, cytc）、半胱氨酸天冬氨酸蛋白酶3（cysteinyl aspartate specific proteinase 3, caspase-3）、半胱氨酸天冬氨酸蛋白酶9（cysteinyl aspartate specific proteinase 9, caspase-9）、Bcl-2以及Bcl-2相关X蛋白（Bcl-2 associated X protein, Bax）的表达水平。
讨论：综上，本研究结果证实，IQC可通过调控Phlpp1/AKT/Bcl-2信号通路改善心肌细胞的线粒体膜通透性，抑制细胞色素c从线粒体内膜释放至细胞质，减少凋亡小体形成，从而拮抗细胞凋亡，减轻THP诱导的心脏毒性。